In this proposal, we seek to unravel the dynamic regulation of histone and non-histone methyl-arginine and citrulline, both at genomic and mechanistic levels. Peptidyl arginine deaminase 4 (hereafter PAD4), is a histone-modifying enzyme that behaves like a transcriptional co-repressor in most instances. PAD4 has been shown to deiminate ('citrullinate') a number of targets, including arginines and methyl-arginines on the N- terminal tails of histones H3 (H3R2, H3R17, and H3R26) and H4 (H4R3), which counteracts the 'activating' methylation of these sites by the coactivators CARM1 and PRMT1. In Aim 1, we develop and describe our system of matched MCF7 breast cancer PAD4 wild type and knockdown cells. Knock down of PAD4 in MCF7 cells results in a cellular motility and invasion, or 'metastatic' phenotype. We will utilize this system in order to identify PAD4 gene targets. Using chromatin immunoprecipitation (ChIP), combined with next-generation high throughput sequencing, we will map PAD4 binding sites across the genome, as well as map sites containing methylated H3 arginine 17 (H3R17me), and H4R3me (PAD4 targets), and histone H3 citrulline 17 (H3Cit17) and H4Cit3 (PAD4 products). We will combine this global ChIP data with our PAD4 gene expression array data in order to produce a complete genomic map of 'true' PAD4 gene targets. Biological functions of this transcriptional network will be examined in light of the metastatic phenotype of PAD4 knockdown cells. In Aim 2, we propose a conceptual advance of the 'histone code hypothesis,' a hybrid between the original histone code and the extensive presence of non-histone post-translational modifications (PTMs). Non-histone proteins contain short stretches of histone sequences, which we have termed histone cassette mimics, and these 'histone cassette mimics' can be post-translationally modified by the same enzymes that target histone sequences. In this aim, describing examples of both candidate-based and unbiased approaches, we will identify non-histone methyl-arginine and citrulline sites in key downstream targets in order to identify important biological substrates that are regulated by post translational modifications and to begin to unravel their roles in cell signaling pathways. Finally, in Aim 3, we will dissect the regulation of PAD4 and citrulline on the protein level, by identifying PAD4 post-translational modifications and protein interactors that recruit PAD4 to chromatin. We will also identify citrulline 'readers', or effector molecules, facilitating the dissection of the downstream signaling events of histone (and nonhistone) citrullination. Detailed insights into the function and regulation of arginine methylation and citrullination on both histone and nonhistone proteins will be key to unraveling pathways involved in normal development and the pathogenesis of human cancers.